Glycerol is a compound in great demand by industry for use in cosmetics, liquid soaps, food, pharmaceuticals, lubricants, anti-freeze solutions, and in numerous other applications. The esters of glycerol are important in the fat and oil industry. Historically, glycerol has been isolated from animal fat and similar sources; however, the process is laborious and inefficient. Microbial production of glycerol is preferred.
Not all organisms have a natural capacity to synthesize glycerol. However, the biological production of glycerol is known for some species of bacteria, algae, and yeast. The bacteria bacillus licheniformis and Lactobacillus lycopersica synthesize glycerol. Glycerol production is found in the halotolerant algae Dunaliella sp. and Asteromonas gracilis for protection against high external salt concentrations (Ben-Amotz et al., (1982) Experientia 38:49-52). Similarly, various osmotolerant yeast synthesize glycerol as a protective measure. Most strains of Saccharomyces produce some glycerol during alcoholic fermentation and this production can be increased by the application of osmotic stress (Albertyn et al., (1994) Mol. Cell. Biol. 14, 4135-4144). Earlier this century glycerol was produced commercially with Saccharomyces cultures to which steering reagents were added such as sulfites or alkalis. Through the formation of an inactive complex, the steering agents block or inhibit the conversion of acetaldehyde to ethanol; thus, excess reducing equivalents (NADH) are available to or “steered” towards dihydroxyacetone phosphate (DHAP) for reduction to produce glycerol. This method is limited by the partial inhibition of yeast growth that is due to the sulfites. This limitation can be partially overcome by the use of alkalis which create excess NADH equivalents by a different mechanism. In this practice, the alkalis initiated a Cannizarro disproportionation to yield ethanol and acetic acid from two equivalents of acetaldehyde. Thus, although production of glycerol is possible from naturally occurring organisms, production is often subject to the need to control osmotic stress of the cultures and the production of sulfites. A method free from these limitations is desirable. Production of glycerol from recombinant organisms containing foreign genes encoding key steps in the glycerol biosynthetic pathway is one possible route to such a method.
A number of the genes involved in the glycerol biosynthetic pathway have been isolated. For example, the gene encoding glycerol-3-phosphate dehydrogenase (DAR1, GPD1) has been cloned and sequenced from Saccharomyces diastaticus (Wang et al., (1994), J. Bact. 176:7091-7095). The DAR1 gene was cloned into a shuttle vector and used to transform E. coli where expression produced active enzyme. Wang et al., supra, recognizes that DAR1 is regulated by the cellular osmotic environment but does not suggest how the gene might be used to enhance glycerol production in a recombinant organism.
Other glycerol-3-phosphate dehydrogenase enzymes have been isolated. For example, sn-glycerol-3-phosphate dehydrogenase has been cloned and sequenced from S. cerevisiae (Larason et al., (1993) Mol. Microbiol., 10:1101). Albertyn et al., (1994) Mol. Cell. Biol., 14:4135) teach the cloning of GPD1 encoding a glycerol-3-phosphate dehydrogenase from S. cerevisiae. Like Wang et al., both Albertyn et al. and Larason et al. recognize the osmo-sensitvity of the regulation of this gene but do not suggest how the gene might be used in the production of glycerol in a recombinant organism.
As with G3PDH, glycerol-3-phosphatase has been isolated from Saccharomyces cerevisiae and the protein identified as being encoded by the GPP1 and GPP2 genes (Norbeck et al., (1996) J. Biol. Chem., 271:13875). Like the genes encoding G3PDH, it appears that GPP2 is osmotically-induced.
Although the genes encoding G3PDH and G3P phosphatase have been isolated, there is no teaching in the art that demonstrates glycerol production from recombinant organisms with G3PDH/G3P phosphatase expressed together or separately. Further, there is no teaching to suggest that efficient glycerol production from any wild-type organism is possible using these two enzyme activities that does not require applying some stress (salt or an osmolyte) to the cell. In fact, the art suggests that G3PDH activities may not affect glycerol production. For example, Eustace ((1987), Can. J. Microbiol., 33:112-117)) teaches hybridized yeast strains that produced glycerol at greater levels than the parent strains. However, Eustace also demonstrates that G3PDH activity remained constant or slightly lower in the hybridized strains as opposed to the wild type.
Glycerol is an industrially useful material. However, other compounds may be derived from the glycerol biosynthetic pathway that also have commercial significance. For example, glycerol-producing organisms may be engineered to produce 1,3-propanediol (U.S. Pat. No. 5,686,276), a monomer having potential utility in the production of polyester fibers and the manufacture of polyurethanes and cyclic compounds. It is known for example that in some organisms, glycerol is converted to 3-hydroxypropionaldehyde and then to 1,3-propanediol through the actions of a dehydratase enzyme and an oxidoreductase enzyme, respectively. Bacterial strains able to produce 1,3-propanediol have been found, for example, in the groups Citrobacter, Clostridium, Enterobacter, Ilyobacter, Klebsiella, Lactobacillus, and Pelobacter. Glycerol dehydratase and diol dehydratase systems are described by Seyfried et al. (1996) J. Bacteriol. 178:5793-5796 and Tobimatsu et al. (1995) J. Biol. Chem. 270:7142-7148, respectively. Recombinant organisms, containing exogenous dehydratase enzyme, that are able to produce 1,3-propanediol have been described (U.S. Pat. No. 5,686,276). Although these organisms produce 1,3-propanediol, it is clear that they would benefit from a system that would minimize glycerol conversion.
There are a number of advantages in engineering a glycerol-producing organism for the production of 1,3-propanediol where conversion of glycerol is minimized. A microorganism capable of efficiently producing glycerol under physiological conditions is industrially desirable, especially when the glycerol itself will be used as a substrate in vivo as part of a more complex catabolic or biosynthetic pathway that could be perturbed by osmotic stress or the addition of steering agents (e.g., the production of 1,3-propanediol). Some attempts at creating glycerol kinase and glycerol dehydrogenase mutants have been made. For example, De Koning et al. (1990) Appl. Microbiol Biotechnol. 32:693-698 report the methanol-dependent production of dihydroxyacetone and glycerol by mutants of the methylotrophic yeast Hansenula polymorpha blocked in dihydroxyacetone kinase and glycerol kinase. Methanol and an additional substrate, required to replenish the xyulose-5-phosphate co-substrate of the assimilation reaction, were used to produce glycerol; however, a dihydroxyacetone reductase (glycerol dehydrogenase) is also required. Similarly, Shaw and Cameron, Book of Abstracts, 211th ACS National Meeting, New Orleans, La., Mar. 24-28 (1996), BIOT-154 Publisher: American Chemical Society, Washington, D.C., investigate the deletion of ldhA (lactate dehydrogenase), glpK (glycerol kinase), and tpiA (triosephosphate isomerase) for the optimization of 1,3-propanediol production. They do not suggest the expression of cloned genes for G3PDH or G3P phosphatase for the production of glycerol or 1,3-propanediol and they do not discuss the impact of glycerol dehydrogenase.
The problem to be solved, therefore, is the lack of a process to direct carbon flux towards glycerol production by the addition or enhancement of certain enzyme activities, especially G3PDH and G3P phosphatase which respectively catalyze the conversion of dihydroxyacetone phosphate (DHAP) to glycerol-3-phosphate (G3P) and then to glycerol. The problem is complicated by the need to control the carbon flux away from glycerol by deletion or decrease of certain enzyme activities, especially glycerol kinase and glycerol dehydrogenase which respectively catalyze the conversion of glycerol plus ATP to G3P and glycerol to dihydroxyacetone (or glyceraldehyde).